One objective of this work is to investigate the area around the active sites of enzymes by the use of bifunctional irreversible inhibitors, so designed that they become chemically or physicaly bound to the active site, and then, already fixed in position, react with another amino acid residue in the vicinity. An inhibitor of this type, which becomes chemically bound to the active site of chymotrypsin, has been found to modify a methionine residue near it. The same methionine residue is also modificed by a series of alkylating agents which are physically bound to the active site by virtue of a benzene ring, which they all contain. Inhibitors of the latter type have been used to label a serine residue at the active site of trypsin and a tyrosine residue at the active site of carboxypeptidase B. Another objective is to probe active sites of enzymes with substrates of restricted conformation. Further research is under way with human thrombin and human plasmin using peptide substrates and reversible and irreversible inhibitors derived from them in order to map the active site, find out why thrombin is so much more specific than trypsin, and possibly develop inhibitors for clinical use.